Alterations in the proliferative/apoptotic equilibrium in semen of adolescents with varicocele

Abstract

Purpose To verify if the presence of varicocele (grades II and III) with and without seminal alterations, using the 5 th centile cutoff values in table A1.1 of the World Health Organization (WHO, 2010) manual, alters the seminal plasma levels of proteins DNASE1 (deoxyribonuclease-1) and IGFBP7 (Insulin-like growth factor-binding protein 7), which are related to apoptosis regulation and cell proliferation, respectively, demonstrating that these proteins are important for correct spermatogenesis. Methods This cross sectional study was performed at the Sao Paulo Federal University Paulo between May 2014 and April 2016. A total of 61 male adolescents were included in this study, of which 20 controls without varicocele (C), 22 with varicocele and normal semen analysis (VNS) and 19 with varicocele and altered semen analysis (VAS). Seminal plasma from each patient was used for Western blotting analysis of individual protein levels. Values of each protein were normalized to a testicular housekeeping protein (PARK7-protein deglycase DJ-1). Results Levels of IGFBP7 protein are increased in varicocele. Levels of DNASE1 are progressively decreased in varicocele (lower in varicocele and normal semen analysis, lowest in varicocele and altered semen analysis) when compared to adolescents without varicocele. DNASE1 levels are positively correlated with sperm concentration and morphology (correlation values of 0.400 and 0.404, respectively; p values of 0.001 and 0.001, respectively). Conclusion In conclusion, in adolescents, seminal plasma levels of IGFBP7, responsible for proliferative activity, are increased in varicocele grades II and III, and DNASE1, responsible for apoptosis regulation, are lower in varicocele, lowest in varicocele and low semen quality. These proteins demonstrate molecular alterations brought upon by varicocele. Moreover, DNASE1 is capable of discriminating a varicocele that causes alterations to semen quality from one that does not. We propose that the initial response of varicocele is to increase proliferative activity which, if followed by regulation of apoptosis, may lead to the ejaculation of a population of sperm that are in accordance with WHO cutoff values but, in the presence of dysregulated apoptosis, leads to lower sperm concentration and morphology.

Cysteine‐rich secretory protein 3: inflammation role in adult varicocoele

“…Following protein detection, membranes were incubated in mild stripping buffer (0.2 mol/L glycine, 0.1% [w:vol] SDS, 1% (vol:vol) Tween 20, pH 2.2) and probed with a primary anti‐human DJ‐1 (Protein deglycase DJ‐1, gene name: PARK7; UniProt accession Q99497) goat polyclonal antibody as loading control protein (SAB2500750, Sigma‐Aldrich, Saint Louis, Missouri, USA) (Belardin et al ., ; Del Giudice et al ., ; Wiśniewski & Mann, ), diluted in blocking buffer (1:1000 [vol:vol]) for 2 h at room temperature under constant agitation. Membranes were washed, incubated with an HRP‐conjugated secondary antibody, and submitted to ECL, as described above.…”

“…In that study, different proteomic pathways were observed and a panel of potential protein biomarkers related to testicular dysfunction in varicocoele was suggested. To confirm the potential function of these proteins as biomarkers, we then performed downstream Western blotting analyses (Belardin et al ., ; Del Giudice et al ., ) which corroborated with the mass spectrometry results.…”

Sperm and Seminal Plasma Proteomics: Molecular Changes Associated with Varicocele-Mediated Male Infertility

“…Acetylation-associated proteins involved in fertilization and acrosome reaction (TALDO1: transaldolase 1, HIST1H2B: histone cluster 1 H2B family mem-ber B, GNPDA1: glucosamine-6-phosphate isomerase 1), apoptosis and DNA damage (HSP90AB1: heat shock protein 90 alpha family class B member 1, PPP5C: protein phosphatase 5 catalytic subunit, RUVBL: RuvBlike proteins), mitochondrial dysfunction and oxidative stress (SDHA: succinate dehydrogenase, PRDX1: peroxiredoxin 1 and GSHR: glutathione reductase) have been proposed as post-translational protein biomarkers in varicocele patients [62]. The list of potential sperm biomarkers in varicocele patients based on fertilization, motility and morphology, DNA damage, oxidative stress, and mitochondrial dysfunction are presented in Table 1 [25,26,54,57,58,[60][61][62][63][64][65][66][67].…”

“…Belardin et al [26] reported that the proliferative or apoptotic equilibrium of seminal plasma is altered in varicocele patients. Insulin-like growth factor-binding protein 7 (IGFBP7) associated with the proliferative process was overexpressed, whereas deoxyribonuclease-1 (DNASE1) involved in the regulation of apoptosis was underexpressed in the seminal plasma of adolescents with varicocele [26]. Furthermore, proteomic analysis by the same group revealed that seminal plasma was enriched with immune response proteins leading to a chronic inflammatory reaction in adolescents with varicocele [26].…”

“…Insulin-like growth factor-binding protein 7 (IGFBP7) associated with the proliferative process was overexpressed, whereas deoxyribonuclease-1 (DNASE1) involved in the regulation of apoptosis was underexpressed in the seminal plasma of adolescents with varicocele [26]. Furthermore, proteomic analysis by the same group revealed that seminal plasma was enriched with immune response proteins leading to a chronic inflammatory reaction in adolescents with varicocele [26]. These changes reflect the alterations in testicular functions leading to decreased semen quality in adolescents with varicocele.…”